The successful function of many systems within multicellular organisms are dependent on cell-cell interactions. Such interactions are affected by the alignment of particular ligands with particular receptors in a manner which allows for ligand-receptor binding and thus a cell-cell adhesion. While protein-protein interactions in cell recognition have been recognized for some time, only recently has the role of carbohydrates in physiologically relevant recognition been widely considered (see Brandley, B. K., and Schnaar, R. L., J. Leuk. Biol. (1986) 40:97; and Sharon, N., and Lis, H., Science (1989) 246:227). Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. Many oligosaccharide structures can be created through the differential activities of a smaller number of glycosyltransferases. Their diverse structures, then, can be generated by transcription of relatively few gene products, suggesting a plausible mechanism for establishing the information necessary to direct a wide range of cell-cell interactions. Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins (lectins) on interacting cells have been described (see Dodd, J., and Jessel, T. M., J. Neurosci. (1985) 5:3278; Regan, L. J., et al., Proc. Natl. Acad. Sci. USA (1986) 83:2248; Constantine-Paton, M., et al., Nature (1986) 324:459; and Tiemeyer, M., et al., J. Biol. Chem. (1989) 263:1671).
A large body of data has been accumulated that implicates a family of receptors, the selectins (or Lectin, EGF, Complement-Cellular Adhesion Molecules) (hereinafter LEC-CAMs) in many of the initial interactions between leukocytes and vascular endothelia. The three known members of this family, L-Selectin (LECAM-1, LAM-1, gp90MEL), E-Selectin (LECAM-2, ELAM-1) and P-Selectin (LECAM-3, GMP-140, PADGEM), each contain a domain with homology to the calcium-dependent lectins (C-lectins), an EGF-like domain, and several complement binding protein-like domains (Bevilacqua et al., Science (1989) 243:1160-1165; Johnston et al., Cell (1989) 56:1033-1044; Lasky et al., Cell (1989) 56:1045-1055; Tedder et al., J. Exp. Med. (1989) 170:123-133).
Identification of the C-lectin domains has led to an intense effort to define carbohydrate ligands for these proteins. There is now general agreement that E-selectin recognizes the carbohydrate sequence NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.1-3)GlcNAc (sialyl-Lewis x, or sLe.sup.x) and related oligosaccharides (Berg et al., J. Biol. Chem. (1991) 265:14869-14872; Lowe et al, Cell (1990) 63:475-484; Phillips et al., Science (1990) 250:1130-1132; Tiemeyer et al., Proc. Natl. Acad. Sci. USA (1991) 88:1138-1142; Tyrrell, Proc. Natl. Acad. Sci. USA, in press). P-Selectin has been reported to recognize the Lewis x structure (Gal.beta.1-4(Fuc.alpha.1-3) GlcNAc) (Larsen et al., Cell (1990) 63:467-474). Others report that an additional terminal linked sialic acid is required for high affinity binding (Moore et al., J. Cell. Biol. (1991) 112:491-499). Recently Polley et al., Proc. Natl. Acad. Sci. USA (1991) 88:6224-6228, have described experiments suggesting that such a structure (sLe.sup.x) is also a ligand for P-Selectin, although there is disagreement on this point.
The carbohydrate ligand for perhaps the most widely studied selectin, L-Selectin, has been extremely difficult to define. This is primarily due to the relative difficulty in obtaining significant quantities of high endothelial venules, the tissue thought to contain most of the native ligand. Data (Imai et al., J. Cell Biol. (1991) 113:1213-1221; Stoolman & Rosen, J. Cell Biol. (1983) 96:722-729; True et al., J. Cell Biol. (1990) 111:2757-2764; Yednock et al., J. Cell Biol. (1987) 104:713-723) suggest the L-Selectin ligand may contain fucose, mannose and/or sialic acid, with possible additional anionic components provided by sulfate or phosphate esters. Recently, glycoprotein ligands of L-Selectin have been isolated from mouse HEV (Imai et al., 1991). These glycoproteins possess many of the residues expected for a native ligand (fucose, sialic acid, sulfate), although neither the structure of the carbohydrate chains nor the exact nature of the residues required for recognition have been defined as yet.
Tumor-associated glycolipids have been reported in fetal tissue and a variety of human cancers, including CML cells (Fukuda, M. N., et al., J. Biol. Chem. (1986) 261:2376; Magnani, J. L., et al., J. Biol. Chem. (1982) 257:14365; Hakomori, S., et al., Biochem. Biophys. Res. Comm. (1983) 113:791). This has led to the hypothesis that these structures may be important in many developmental and oncogenic processes (Magnani, J. L., et al., J. Biol. Chem. (1982) 257:14365). Smaller quantities of most of these carbohydrates can be found in normal human tissue (see Fukushi, Y., et al., J. Exp. Med. (1984) 160:506), but until now no function for these structures has been reported.
Adhesion of circulating neutrophils to stimulated vascular endothelium is a primary event of the inflammatory response.
LECAM-1 is particularly interesting because of its ability to block neutrophil influx (Watson et al., Nature (1991) 349:164-167). It is expressed in chronic lymphocytic leukemia cells which bind to HEV (see Spertini et al., Nature (1991) 349:691-694). It is also believed that HEV structures at sites of chronic inflammation are associated with the symptoms of diseases such as rheumatoid arthritis, psoriasis, and multiple sclerosis.
The present inventors have now found that selectins recognize derivatives of triterpenoid acids which can inhibit binding between leukocytes and endothelial cells and, as a consequence of that discovery, have developed the present invention.